A study of genomic variation in and the development of detection techniques for potato virus Y in South Africa

[摘要] ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry.The incidence of this virus has greatly increased over the past few years. Even more worrying is thevariation of symptoms observed during PVY infection and the recent appearance of the more virulentPVYNTN strain in local fields. This project aimed to investigate the possible genetic variation within theviral genome and to establish the origin of strains. The project also aimed to establish a dependable, areaspecific enzyme-linked immunosorbent assay (ELISA) to replace the currently used ELISAs. Currentlyseed potato certification is done using ELISA kits imported from Europe. These kits were developed forthe detection of overseas variants of PVY and the use thereof in South Africa has in the past lead to falsenegatives. Finally, this project set out to develop, optimize and establish a sensitive and reliable real-timereverse transcriptase polymerase chain reaction (qRT-PCR) detection protocol for PVY.In the first part of the study the coat protein (CP) gene of PVY isolates from plant material obtained fromvarious parts of South Africa was amplified using RT-PCR. The resulting cDNA was then sequenceddirectly or cloned into a vector and then sequenced. The resulting sequences were aligned in a data matrixwith international reference sequences, analyzed and grouped according to strain. Examination of the CPgene within this matrix as well as phylogenetic analysis revealed six main groups of PVY. These sixgroups included the traditional PVYN and PVYO groups and a recombinant group. Furthermore it alsorevealed variants of PVYN and PVYO. These mutants and recombinants pose a threat as they may lead toSouth African strains of PVY expressing coat proteins which vary from those found overseas. This mayrender the currently used European ELISA method of detection less effective and subsequently result inan increase in viral prevalence. This reinforced the need for a detection method based on local viralstrains. Phylogenetic and Simplot analysis also confirmed that a recombinant strain between PVYN andPVYO had evolved and that PVYNTN was such a recombinant.The second part of the study aimed to develop and establish detection methods based on local variants ofPVY. This included the development of ELISA and qRT-PCR detection methods of PVY. Previouslyamplified cDNA of the PVY CP gene was cloned into an expression vector and successfully expressed.Antibodies produced against the recombinant protein, when used in ELISA, however, failed to achievethe required levels of sensitivity. This prompted the development of qRT-PCR detection methods forPVY. Primer combinations for PVY were designed using the previously established CP gene data matrix.A reliable and sensitive SYBR® Green I based qRT-PCR assay was developed for the detection of PVY.The assay effectively detected all known South African variants of PVY. Furthermore, a Taqman® assaywas developed for the detection of all variants of PVY. The Taqman® assay was 10 fold less sensitive anddoes not allow for amplicon verification through melting curve analysis, but it does add more specificitydue to the addition of the probe. Although these qRT-PCR detection methods are still too expensive toreplace the routine diagnostics done with ELISA, they do offer the opportunity to screen valuable mothermaterial and confirm borderline cases in seed certification.

[发布机构] Stellenbosch University